Roles of Protein Post-Translational Modifications During Adipocyte Senescence

Adipocytes are adipose tissues that supply energy to the body through lipids. The two main types of adipocytes comprise white adipocytes (WAT) that store energy, and brown adipocytes (BAT), which generate heat by burning stored fat (thermogenesis). Emerging evidence indicates that dysregulated adipocyte senescence may disrupt metabolic homeostasis, leading to various diseases and aging. Adipocytes undergo senescence via irreversible cell-cycle arrest in response to DNA damage, oxidative stress, telomere dysfunction, or adipocyte over-expansion upon chronic lipid accumulation. The amount of detectable BAT decreases with age. Activation of cell cycle regulators and dysregulation of adipogenesis-regulating factors may constitute a molecular mechanism that accelerates adipocyte senescence. To better understand the regulation of adipocyte senescence, the effects of post-translational modifications (PTMs), is essential for clarifying the activity and stability of these proteins. PTMs are covalent enzymatic protein modifications introduced following protein biosynthesis, such as phosphorylation, acetylation, ubiquitination, or glycosylation. Determining the contribution of PTMs to adipocyte senescence may identify new therapeutic targets for the regulation of adipocyte senescence. In this review, we discuss a conceptual case in which PTMs regulate adipocyte senescence and explain the mechanisms underlying protein regulation, which may lead to the development of effective strategies to combat metabolic diseases.


Introduction
Adipogenesis is a key process during cell differentiation from pre-adipocytes to mature adipocytes.Adipocytes proliferate to form adipose tissue, where the energy harvested from food that exceeds the energy expenditure is stored as lipids.Pre-adipocytes develop in four stages: growth arrest, mitotic clonal expansion, early differentiation, and terminal differentiation into mature adipocytes [1].In this process, pre-adipocytes accumulate lipids by increasing the quantities of enzymes needed for triglyceride (TG) synthesis and the accumulation of TG.Increased expression of transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), and CCAAT/enhancer binding protein β (C/EBPβ), is essential for adipocyte differentiation [2] (Figure 1A).PPARγ is a significant regulator of adipogenesis, and the C/EBP family (α and β) is one of the most critical downstream targets of PPARγ.These proteins are also necessary for the transcription and expression of insulin receptors, adiponectin, adipocyte protein 2 (aP2), and adipokines in mature adipocytes [3,4].
Understanding the processes involved in adipocyte senescence and their regulation is essential.Adipocyte senescence represents an irreversible cell Ivyspring International Publisher cycle arrest in response to various stressors, including DNA damage, oxidative stress, metabolic stress, and telomere shortening [5,6] (Figure 1B).These stressors transmit signals through multiple pathways, most of which activate the cell cycle inhibitor p53.These pathways converge upon the activation of the cyclin-dependent kinase (CDK) inhibitors p16, p21, p27, and p15.Eventually, they activate retinoblastoma protein (RB), causing senescence [7].Unrepaired DNA damage and the loss of repair capacity can induce senescence [8].In adipose tissue, p53 contributes to insulin resistance in age-associated metabolic diseases.Excessive caloric intake exacerbates oxidative stress and increases the production of p53 and pro-inflammatory cytokines in adipose tissue.In contrast, p53 suppression in adipose tissue ameliorates senescence-like changes by reducing proinflammatory cytokines and improving insulin sensitivity [9].Interestingly, PPARγ controls adipocyte differentiation, inhibits cellular proliferation, and promotes cellular senescence [10,11].PPARγ induces the expression of p16 INK4α (CDKN2A), a cell cycle inhibitor that promotes senescence, increases senescence-associated-βgalactosidase (SA-β-gal) levels, and triggers G1 arrest [11].Cellular senescence causes functional disorders of adipogenesis and lipid storage in adipose-derived stromal/progenitor cells [12] (Figure 1C).
Browning (or beiging) of adipose tissue refers to the conversion of white adipocytes (WAT) into brown-like adipocytes, such as beige or brite cells (Figure 2).Brown adipocytes (BAT) are thermogenic, meaning they produce heat by burning stored fat.Adipose tissue browning is typically associated with increased energy expenditure and improved metabolic health.BAT, which are responsible for energy production, contain more mitochondria than do WAT which provide energy storage [13].Developmentally, in mice, BAT originates from a myogenic factor 5 (MYF5)-positive mesoderm lineage [14] (Figure 2A).Transcriptional control of the BAT-specific thermogenic program is mediated by PPAR-γ coactivator 1-alpha (PGC-1α) and PR domain-containing 16 (PRDM16) [15] (Figure 2B).It is well established that BAT rely on mitochondrial function for maintaining intracellular metabolism.In addition, BAT mitochondria are functionalized by uncoupling protein-1 (UCP1), which allows the translocation of protons to dissipate energy during non-shivering thermogenesis [16,17] (Figure 2B).Forkhead box protein A3 (FOXA3) expression is increased in visceral fat during aging and has been reported to reduce BAT mass and the beiging capacity of WAT [18,19].In the context of aging, it has been reported that mitochondrial enzyme expression is reduced in adipose tissue from old mice, yet little is known regarding the mechanisms that mediate these changes [20,21].Similarly, it is well-established that human WAT mitochondrial function, as measured by tissue oxygen consumption, is reduced in both obesity and aging [21].Consistent with this, Foxa3-knockout mice are long-lived, have increased BAT activity late in life, and are protected from age-related insulin resistance and high-fat diet-induced increases in visceral adiposity [19] (Figure 2C).
Evidence suggests that replicative capacity and UCP1 expression in BAT are significantly reduced during aging [16,17].In particular, the proliferative capacity and UCP1 expression in response to cold stimuli appear to be abolished in aged BAT [22].Increasing age of brite adipocytes progressively leads to the development of a WAT phenotype, which prevents adipocyte browning in older mice and humans [23,24].Conversely, the levels of the senescence markers p16 and p21 are highly increased during the senescence process in BAT [13,25].BAT thermogenesis declines with aging [26] (Figure 2D), consistent with the reduction in the thermogenic factor UCP1, whereas UCP1 levels in BAT are stabilized by SIRT5 desuccinylation.Sirt5 deficiency in BAT increases the succinylation of UCP1, resulting in decreased UCP1 stability and function, which impairs mitochondrial homeostasis and alters BAT-mediated thermogenesis [27].This may thus be one of the mechanisms regulating BAT senescence.
The senescence-associated secretory phenotype (SASP), which includes pro-inflammatory cytokines, is produced via autocrine and paracrine pathways to enhance and diffuse senescent cell influence and induce chronic inflammation in adipose tissues [29].Under stress stimulation conditions, these interactive signaling pathways converge toward the activation of a transcriptional program managed by nuclear factor kappa B (NF-κB) and C/EBPβ, the core effectors that initiate and maintain SASP gene expression [30].
Removing senescent adipocytes mitigates inflammation and ameliorates insulin resistance in adipose tissue [29].Therefore, the proper control of adipocyte senescence is critical for the management of various diseases.The mechanisms controlling cellular senescence can be divided into three categories.These include transcriptional modifications, such as DNA methylation at the genetic and epigenetic levels; mRNA-level regulatory mechanisms, such as mRNA stabilization or degradation; and post-translational modifications (PTMs), such as ubiquitination [31].Here, we focused on PTMs that regulate protein stability and activation.PTMs such as phosphorylation, acetylation, ubiquitination, and glycosylation alter the chemical properties and functions of proteins [32].Abnormal PTMs can cause biological dysfunction and lead to various diseases.For example, PTMs significantly affect the structure and function of proteins that regulate adipocyte senescence.Understanding the role of PTMs in adipocyte senescence may guide the discovery of new therapeutic targets to modulate adipocyte senescence.
In this review, we summarize how the stability and activation of proteins involved in the induction of adipocyte senescence are regulated by various PTMs (Table 1) and provide new insights regarding the regulation of adipocyte senescence (Figure 3).

Regulation of protein activation by phosphorylation
Adipocyte senescence is tightly regulated to maintain energy and metabolic homeostasis [26].Senescent cells accumulate in aging fat in response to replicative, cytokine-induced, and metabolic stresses [33].Janus kinase (JAK), p38, and the phosphatase and tensin homolog on chromosome 10 (PTEN) upregulate adipocyte senescence under various cellular stress conditions.Cellular phosphorylation is known to potentiate or downregulate adipocyte senescence; however, the underlying mechanisms require further investigation.

JAK
An increase in cellular senescence in response to intrinsic or extrinsic stresses and the broadly related SASP promote organismal aging and adipose tissue dysfunction [26,34,35].Inhibition of the JAK/signal transducer and activator of transcription (JAK/STAT) pathway, which plays a significant role in adipose tissue development and function and regulates SASP, can partially inhibit SASP secretion [34,36].The JAK1/2 inhibitor ruxolitinib decreases the pro-inflammatory SASP in vitro and in vivo and enhances insulin sensitivity in aging mice [34].Aging cell improvement enhances adipogenesis and metabolism [37,38].Activin A secreted by senescent cells, blocks adipogenesis.Treatment of aged mice and primary human senescent fat progenitor cells with a JAK inhibitor reduced activin A levels and restored lipid accumulation and expression of critical adipogenic markers.JAK inhibitors also reduce lipotoxicity and increase insulin sensitivity [37].The inhibition of JAK activity is a strategy used to alleviate adipocyte cellular senescence by ameliorating senescent adipose progenitors and stem cells (APSCs).

PTEN
The insulin signaling response displays heightened sensitivity to cellular aging in adipocytes, thereby decreasing the insulin response [6,26,43,44].PTEN is a phosphatidylinositol phosphate phosphatase that plays an essential role in various cellular processes, including genome maintenance, DNA repair, cell cycle control, proliferation, metabolism, migration, tumorigenesis, and senescence.One of the critical roles of PTEN is to inhibit insulin signaling [45][46][47][48].PTEN negatively regulates insulin signaling by dephosphorylating phosphatidylinositol-3,4,5triphosphate (PIP3), resulting in decreased phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling [49].Pten deficiency enhances energy expenditure in brown adipose tissue.PTEN loss and activation of PI3K/AKT signaling lead to an improved ability to handle metabolic stress in mice [50].
PTEN downregulation increased the proliferation of stromal vascular fraction (SVF) cells, including adipocyte progenitor cells, in adipose tissues.Pten deficiency restores the differentiation capacity of high-passage SVF cells and increases adipogenesis.In contrast, PTEN expression is upregulated during cellular senescence [51].The regulation of PTEN activity and expression levels is a strategy for controlling adipocyte cellular senescence.

Regulation of protein activation by deacetylation
Senescent adipocytes accumulate in aging fat in response to cytokines and metabolic stress [26,33].Adipocyte senescence is regulated in response to cold exposure-stimulated thermogenesis.The mitochondrial function and activity of UCP-1, a thermogenesisrelated mitochondrial protein in adipocytes, decrease with cellular aging.Additionally, the pro-inflammatory capacity of BAT increases with age [26,35,52].Histone deacetylases (HDACs) such as sirtuin 1 (SIRT1), SIRT 6, and HDAC1 are essential regulators of adipocyte senescence under conditions of cellular stress [26,53,54].Although SIRTs and HDAC1 are deacetylases, their roles in adipocyte senescence differ.SIRT1 and SIRT6 prevent adipocyte senescence, whereas HDAC1 potentiates adipocyte senescence.The number of brown and beige adipocytes decreases with age.Cellular deacetylation events regulate adipocyte senescence.However, the underlying mechanisms remain unclear.

SIRT6
The essential roles of SIRT6 in adipocytes are the regulation of lipid metabolism and the prevention of inflammation.SIRT6 stimulates lipolysis, enhances adipose tissue browning, and ameliorates adipose tissue inflammation, thereby improving insulin action in the peripheral tissues [63].SIRT6 promotes cell proliferation and antagonizes cellular senescence; however, SIRT6 expression decreases during cellular senescence [64].Moreover, SIRT6 suppresses p27 Kip1 (p27) expression during cellular senescence [64].SIRT6 mediates the polyubiquitination of p27, directing its degradation by the proteasome and thereby regulating the acetylation status of p27.Thus, SIRT6 delays cellular senescence [64].Aging and excessive caloric intake, which are two major risk factors for obesity and diabetes, lead to decreased SIRT6 levels [65].Sirt6 deficiency in pre-adipocytes blocks adipogenesis and regulates mitotic clonal expansion [66].Sirt6 deletion in adipose tissue impairs the thermogenic function of BAT, causing morphological ''whitening'' of brown fat, reduced oxygen consumption, obesity, decreased core body temperature, and cold sensitivity.Fat Sirt6-deleted mice exhibit increased blood glucose levels, severe insulin resistance, and hepatic steatosis.Moreover, Sirt6 deficiency inhibits WAT browning following cold exposure or β3-agonist treatment [67].Taken together, these results indicate that SIRT6 plays a protective role against adipocyte senescence.

Regulation of protein stability by ubiquitination: E3 ubiquitin ligases
Another regulatory mechanism that influences physiological cellular senescence is the post-translational modification of cellular proteins through ubiquitination.The ubiquitin-proteasome pathway (UPP) regulates the differentiation of various cell types.Alterations in the UPP in mature adipocytes can potentially modulate adipose function during adipocyte aging [75].Ubiquitin is activated by E1 and transferred to E2 or Ub conjugase.In turn, the E2 enzyme transfers ubiquitin to E3 or directly ubiquitinates the target protein in conjunction with E3 [72,73].Proteasome activity decreases during senescence, which may be associated with aging and age-associated diseases [75].E3 ubiquitin-protein ligases are crucial factors in the regulation of senescence by ubiquitination.Knockdown of regulator of cullins-1 (ROC1), a component of the SKP, Cullin, F-box (SCF) E3 ubiquitin ligases, suppresses the growth of several human cell lines by inducing senescence [76].In addition, the inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, which encodes a subunit of an E3 ubiquitin ligase, causes a senescence-like phenotype in human cancer cell lines [77].Therefore, E3 ubiquitin ligase activity appears to be essential for regulating adipocyte senescence.Several E3 ligases have recently been found to be present in adipocytes, including seven in absentia homolog 2 (SIAH2), makorin ring finger protein 1 (MKRN1), tripartite motif protein 23 (TRIM23), and neural precursor cell-expressed developmentally downregulated protein 4 (NEDD4) [78].The ubiquitination pathway regulates p53 tumor suppressor stability, localization, and functions in normal cells [79].E3 ubiquitin ligases, including murine double minute 2 (MDM2) and MKRN1, predominantly regulate p53 expression levels and activities under various physiological conditions [79,80].Overexpression of MDM2 and CDK4 can induce human telomere reverse transcriptase (hTERT) overexpression and p53 degradation in human 2H transgenic bone marrow-mesenchymal stem cells (BM-MSCs), increase cell proliferation and migration, and suppress the adipogenic differentiation potential in vitro [81].

Regulation of protein activation by O-GlcNAcylation
O-linked-N-acetylglucosamination (O-GlcNAcylation), a post-translational glycosylation event, occurs on proteins in the nucleus, cytoplasm, and mitochondria, regulates cell signaling, and is associated with several pathological conditions [96].O-GlcNAcylation is the single-sugar addition of O-linked-β-N-acetylglucosamine (O-GlcNAc) to the hydroxyl groups of the serine or threonine residues of target proteins [97].The attachment and removal of O-GlcNAc from proteins are processed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) [98].O-GlcNAcylation is highly responsive to glucose, and insulin resistance enhances O-GlcNAcylation [99].Increased levels of O-GlcNAcylation have been observed in response to the induction of adipocyte differentiation [100].Post-translational modifications regulate OGT activity, and OGT activation occurs when tyrosine phosphorylation of OGT increases following insulin stimulation in 3T3-L1 cells [101].Abnormal protein O-GlcNAcylation is essential for the development and progression of senescencerelated diseases [102].
In 3T3-L1 adipocytes, O-GlcNAc causes post-transcriptional modification of PPARγ.The primary O-GlcNAc site of PPARγ is threonine 54 of the N-terminal activation function-1 domain [97].In 3T3-L1 cells, an increase in O-GlcNAc modification mediated by the OGA inhibitor NButGT decreases PPARγ transcriptional activity and terminal adipocyte differentiation [97].
C/EBPβ is also modified by O-GlcNAc, which is present in nucleocytoplasmic proteins.GlcNAcylation sites (Ser 180 and Ser 181 ) are located in the regulatory domain and are extremely close to the phosphorylation sites (Thr 188 , Ser 184 , and Thr 179 ) that regulate DNA-binding activity [102].GlcNAcylation of Ser 180 and Ser 181 blocks the phosphorylation of Thr 188 , Ser 184 , and Thr 179 , thereby delaying adipocyte differentiation [102].In contrast, the mutation of Ser 180 and Ser 181 to Ala increased C/EBPβ transcriptional activity [102].Therefore, GlcNAcylation and phosphorylation appear to modulate the function of C/EBPβ by alternately occupying adjacent sites.
The hexosamine biosynthesis pathway (HBP) flux functions as a nutrient sensor and induces O-GlcNAc modification of the AMPK α subunit in both immortal and primary murine adipocytes [103].O-linked glycosylation via HBP flux regulates AMPK activation and induces fatty acid oxidation in 3T3-L1 adipocytes [104] whereas removal of O-GlcNAc by hexosaminidase reduces AMPK activity [104].Thus, HBP correlates with O-GlcNAc and is likely to affect adipocyte senescence.

Conclusions and perspectives
Adipogenesis is the process of differentiation of pre-adipocytes into mature adipocytes.There are two main types of fat cells that contain lipids: WAT, which store energy, and BAT, which produces heat.Pre-adipocytes develop in four stages: growth arrest, mitotic clone expansion, early differentiation, and terminal differentiation into mature adipocytes [1].In this process, increased expression of transcription factors is essential for adipocyte differentiation [2] (Figure 1A).
Upon DNA damage, oxidative stress, metabolic stress, or telomere shortening, adipocytes undergo senescence and irreversible cell cycle arrest, thereby inhibiting adipogenesis [5,6].These cellular stressors activate p53 and induce CDK inhibitors and RB, which cause senescence [7].In addition, the modification of adipogenesis regulators such as PPAR and C/EBPα/β results in adipocyte senescence.Moreover, mitochondrial UCP1 expression has been reported to decrease in old mouse adipocytes; however, little is known about this mechanism [16,17,27].As UCP1 levels decrease during the aging of BAT, the levels of aging markers p16 and p21 increase significantly [13,25].In addition, the expression of UCP1, a thermogenesis factor, in BAT is stabilized by SIRT5 desuccinylation.This may thus be a mechanism by which BAT senescence is regulated; however, this possibility requires further investigation.
Once lipids continuously accumulate in the adipose tissue, the adipocyte senescence rate increases and insulin sensitivity decreases, resulting in adipose tissue dysfunction.SASP induces chronic inflammation in adipose tissue.Cellular senescence causes lipid storage dysfunction.Thus, the appropriate control of fat cell aging is a viable strategy for preventing aging-related diseases.Although the underlying mechanisms remain poorly understood, adipocyte senescence is essential for diverse physiological processes, including metabolism and various age-related diseases.To better understand the processes of adipocyte senescence, it is important to identify the modulators of adipogenic factors, including PPARγ, and their regulatory molecular mechanisms, such as PTMs.PTMs are associated with oxidative stress, inflammation, and aging, thereby influencing aging characteristics [105].Some PTMs participate in healthy aging, suggesting that they are essential regulators and predictive markers of the senescence process [106].PTMs significantly affect aging by targeting epigenetic and non-epigenetic pathways.Therefore, understanding the role of PTMs in cellular senescence may advance the development of targeted therapies for age-related diseases.p38, JAK, and PTEN influenced fat cell senescence through phosphorylation under stress (Figure 3).Inhibition of the JAK/STAT pathway inhibits SASP secretion and Pten deficiency increases adipogenesis [34,[36][37][38][39].
O-GlcNacylation regulates cell signaling and plays an essential role in the development and progression of age-related diseases.OGT and OGA regulate the attachment and removal of O-GlcNAc.In 3T3-L1 cells, these enzymes induce adipocyte senescence.We predict that new treatments for adipocyte senescence can be developed by understanding the involvement of PTMs in fat-cell aging (Figure 3).
In particular, a clearer understanding is needed regarding how PTMs, including acetylation/deacetylation, phosphorylation, ubiquitination, and glycosylation, regulate the function of necessary adipogenic factors, such as PPAR (Figure 3).Finally, it is important to establish the degree to which prevention of adipocyte senescence mediates beneficial effects on adipose tissue in various disease conditions.Future research on the regulatory mechanisms underlying adipocyte senescence will likely provide critical insights regarding the new and complex networks involved in human biological processes, including aging and metabolism.

Figure 1 .
Figure 1.Overview of differentiation and cellular senescence in adipocytes.(A) Adipogenesis, (B) senescence, and (C) induction of disease.Pre-adipocytes differentiate into mature adipocytes during adipogenesis.PPARγ and C/EBPα/β are regulators of adipogenesis, and their expression is increased.Also, insulin receptors, adiponectin, aP2, and adipokines are highly expressed in mature adipocytes.Stressors such as hyper-proliferation, DNA damage, oxidative stress, metabolic stress, and telomere shortening activate p53, CDK inhibitors, and RB, which induce senescence.During the accumulation of senescent cells, cell size increases, and free fatty acids (FFAs) are released at high levels with increased inflammation and collagen.Decreased adiponectin and poor insulin sensitivity also occur.These factors are related to cellular dysfunction and cause various diseases such as obesity, diabetes, chronic inflammation, and fibrosis.

Figure 2 .
Figure 2. Processes deciding adipocyte fate during differentiation and senescence, and their regulators in adipose tissues.Adipose tissue can be divided into white adipose tissue (WAT) and brown adipose tissue (BAT).(A) Through the differentiation of adipocytes, WAT and BAT develop from precursor cells, and Myf5 is expressed from the precursor cells.Pre-adipocytes turn into WAT and BAT during maturation, with transcription factors such as PPARγ, C/EBPs, and PRDM16 regulating this process.Browning of the adipose tissue leads to increased energy expenditure and improved metabolic health.Compared with WAT, BAT contains more mitochondria, which are related to energy production.The increased expression of PPARγ, C/EBPγ, and C/EBPγ is associated with WAT differentiation.(B) BAT is regulated by PGC-1α and PRDM16.Additionally, BAT mitochondria are functionalized by UCP1.With increased lipotoxicity, mitochondrial dysfunction, ROS, and DNA damage, adipocytes become senescent.(C) During senescence, FOXA3 expression is increased concomitant with reduced BAT mass and beiging capacity of WAT.(d) During BAT senescence, mitochondrial enzyme expression is reduced, as is UCP1 expression.In contrast, the expression levels of p16 and p21 are increased.